Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a stroke disorder caused by mutations in Notch3. Hallmark pathological features of CADASIL include: rarefication of the extracellular matrix, granular osmiophilic material (GOM) deposition, and Notch3 protein accumulation. Identification of molecular cascades that lead to these abnormalities will potentially lead to therapies which can prevent progression of this debilitating disease. Normal Notch signaling requires trans-endocytosis of the Notch3 ectodomain, resulting in movement of Notch from one cell to another. This process reduces Notch3 levels. We propose that defects in trans-endocytosis result all of the hallmark pathological features of CADASIL. We have recently identified a family of proteins that interacts with Notch3 and participate in trans-endocytosis of Notch3. Preliminary data demonstrates specific physical interactions between Notch3 and LRP1, a protein known for its endocytic function. Our data indicate that LRP1 plays an important role in trans- endocytosis of Notch3, which potentiates Notch3 function. Based on these findings, we suggest the following hypothesis: mutant Notch3 in CADASIL dysfunctionally binds to LRP1, leading to LRP1 malfunction; decreased LRP1 results in inhibition of endocytosis of critical extracellular proteins, including Notch3. We propose to test this hypothesis in three specific aims. First, we will determine at the molecular level whether mutant Notch3 proteins interact differently with LRP1 (compared to WT Notch3). Second, we will determine in cell cultures whether mutant Notch3 inhibits LRP1 dependent endocytosis. Third, we will test our overall hypothesis by examining mice with tissue-specific inactivation of LRP1 to test whether LRP1 is a true target of mutant Notch3 in vivo. These studies may lead to important directions in the treatment of CADASIL, since our main hypothesis indicates that targeting the interaction between mutant Notch3 and LRP1 may slow the disease process. In addition, recent evidence shows that LRP1 participates in vascular dysfunction after stroke; our studies may thus offer additional insights into the mechanisms of how LRP1 in the brain regulates vascular homeostasis. In lay termninology, this proposal will define the precise mechanism of how Notch proteins can be regulated by LRP1. We will determine if CADASIL Notch3 mutants influence LRP1 function, and whether LRP1 dysfunction results in the vascular pathology seen in CADASIL patients.